PROVIPED. t The function of heat shock proteins (HSP) is to chaperone intracellular proteins and peptides generated during protein synthesis and degradation. This includes proteins and peptides encoded and generated by infectious agents and tumor antigens. The immune system has evolved a sophisticated system to monitor necrotic cell death by detecting cell released HSPs via heat shock protein receptors on APC, and to screen HSPs for chaperoned antigenic peptides by cross presenting them to CDS cells. By generation of a secreted form of hsp-gp96 we created a model imitating cell damage and a system for analyzing in vivo the biological effects of hsp-gp96. This.was done by replacing the KDEL ER-retention signal of human gp96 with the Fc portion of lgG1 to generate a fusion protein, gp96-lg. Gp96-lg-transfected tumor cells secrete gp96-lg in vivo and mediate strong, cognate CD8-CTL expansion, cause tumor rejection and generate long term anti-tumor immunity. We show that secreted gp96-lg-associated peptides are approximately one million fold more efficient than native protein in mediating cognate CDS cross-priming. We also show that tumors can orchestrate tolerogenicity via induction of tolerizing chemokines in their micro environment. Secretion of gp96-lg by tumors triggers a switch in chemokine production in the micro- environment from tolerogenic (induced by w.t. tumors) to immunogenic chemokines (induced by gp96- secretion) resulting in DC and NK recruitment and activation. In specific aim 1 we will analyze which cells in the microenvironment execute this chemokine switch and test the hypothesis that this switch is required for immunogenicity of gp96. B cells inhibit tumor immunity and gp96 induced CDS clonal expansion. In collaboration with project 3, we will study the mechanisms of B cell inhibition of the CDS response. Together with project 2 we will examine how CDS memory cells are regulated by tumor secreted gp96-lg. In aim 2 we will use the gp96-lg model system to dissect the mechanisms by which established tumors induce immune tolerance and how tolerance can be reversed. We will test the hypothesis that established tumors induce tolerogenic chemokines in their microenvironment.and that the blockade of these chemokines will synergize with tumor rejection by gp96-vaccines. We will also study the effect of B cell depletion in combination with gp96-immunization after bone marrow transplantation, in collaboration with project 3 and 4. We have generated initial data supporting a new paradigm in immunotherapy: Non-immunogenic tumors are ideal targets for vaccine therapy. In specific aim 3 we plan to further test this hypothesis in patients with terminal non-small cell lung carcinoma (NSCLC). We propose and will test that gp96 vaccines have ideal vaccine properties due to enhancing antigen cross presentation to CDS cells, activation of innate immunity, and creating a Th1 environment.